Novel substituted 7-acetylamino cephalosporanic acids



United States Patent ABSTRACT OF THE DISCLOSURE This disclosuredescribes compounds of the class of 7-(azialkanoylamino)cephalosporanicacids, 7-[(a-phenyl)azialkanoylamino]cephalosporanic acids,7-[(w-carboxy)azialkanoylamino]cephalosporanic acids and 7- [(a phenyl wcarboxyl)azialkanoylamino]cephalosporanic acids; useful asanti-bacterial agents.

This invention relates to new derivatives of 7-aminocephalosporanic acidand, more particularly, is concerned with novel compounds which may berepresented by the following general formula:

wherein m has a value of 0, 1 or 2; nhas a value of 0, 1 or 2; A isacetoxy or N-pyridinium; M is hydrogen, pharmaceutically acceptablenontoxic cations or an anionic charge when A is N-py-ridinium; Q ishydrogen or carboxy; and W is hydrogen or phenyl with the proviso thatwhen W is phenyl then It must not be 0.

DETAILED DESCRIPTION OF THE INVENTION In general Formula I set forthabove, suitable azialkanoyl groups contemplated by the present inventionmay be, for example, 3-azibutanoyl, 4-azipentanoyl, 4-azi-2-phenylpentanoyl, 5 azihex-anoyl, 4-azi-6-carb0xyhexanoyl,S-azioctanoyl, S-azi-S-carboxyoctanoyl, S-azi- 2-pheny1octanoyl,S-azi-Z-phenyl-S-carboxyoctanoyl, and the like. In those instances WhereA is N-pyridinium, the cationic charge on this group is matched by theanionic charge of the carboxylic acid radical, the entire molecule beingof a zwitterion nature and m is thus an anionic charge.

Also embraced within the scope of the present invention are thenontoxic, pharmaceutically acceptable salts of these derivatives of7-aminocephalosporanic acid. Included are the monobasic salts when Q ishydrogen and the dibasic salts when Q is carboxy. The cations comprisedin these salts and embraced by M include, for example, the nontoxicmetal cations such as the sodium ion, potassium ion, calcium ion,magnesium ion as well as the organic amine cations, such as thetri(lower alkyl) amine cations (e.g., triethylamine), procaine, and thelike.

The novel compounds of the present invention, when A is acetoxy inFormula I above, may be readily prepared by acylating7-aminocephalosphoranic acid with a compound of the formula:

N=N W (II) wherein m, n, Q and W are as hereinbefore defined and Z is ahalide (preferably chloride), azide, acyloxy or p- Patented Oct. 3, 1967ice nitrophenoxy group. This acylation of the 7-arninocephalosporanicacid is performed, for example, by the Schotten-Baumann method, takinginto consideration the sensitivity of these compounds, under mildconditions and advantageously in the presence of a diluent or solventsuch as water or an organic solvent, for example, a ketone such asacetone, an ether such as tetrahydrofuran, or a halogenated hydrocarbonsuch as chloroform or methylene chloride. The reaction is preferablyconducted in the presence of a basic agent such as sodium bicarbonate orpotassium bicarbonate, or an organic base such as one of the organicbases listed hereinbefore. The reaction is also preferably carried outat a temperature of from about 0 C. to about 25 0., preferably at 0 C5C.,

and over a period of time of afew hours or more. Where Q is carboxy, thediacid halide, azide or ester is condensed with 7-aminocephalosporanicacid under conditions to give monoacylation and then hydrolyzed duringthe workup and separation of isomers to give the desired product. Theremoval of other isomers, when formed in appreciable amounts, m'aybe'readily carried out by chromatographic or crystallization procedures.

The acylating agents cor-responding to Formula II, when new, may beprepared by methods well-known in the art from the corresponding acids(Z is hydroxy). Thus, the acid can be treated with thionyl chloride oroxalyl chloride, if desired, in the presence of dimethylformamide, toyield the corresponding acyl chlorides (Z is chlorine), which, ifdesired, can be converted to the acyl azides (Z is N by treatment withsodium azide. The p-nitrophenyl esters (Z is p-nitrophenoxy) can beprepared by following the procedure of Bodanszky et a1. (BiochemicalPreparations,vvol. 9, p. 110, .1962, John Wiley and Sons, New York,N.Y.). Specific acylating agents operable in this process (where Q ishydrogen) include, for example, 3-az'ibutanoyl chloride, 4-azipentanoylazide, 4-azi-2-phenylpentanoyl bromide, p-. nitrophenyl S-azihexanoate,S-azioctanoyl bromide, S-azi- 2-phenyloctanoyl chloride, and the like.Specific acylating agents operable in this process (where Q is carboxy)include, for example, 4-azi-6-carboxyhexanoic acid di-pnitrophenylester, 5-azi-8-carboxyoctanoic acid diazide,S-azi-Z-phenyl-S-carboxyoctanoic acid diacid chloride, and the like. Theintermediate azialkanoic acids (Z is bydroxy) corresponding to FormulaII above may be prepared from the corresponding ketoalkanoic acids bytreatment with hydroxylamine-O-sulfonic acid after conversion of theketoalkanoic acid to the imine with liquid amonia.

Thenovel compounds of the present invention, when A is N-pyridinium inFormula I above, may be readily prepared from the corresponding7-aminocephalosporanic acid derivatives (A is acetoxy in Formula Iabove) by treatment with pyridine in water or aqueous acetone at 20-50C. and for a period of time of about 1-3 days. The resulting3-.(l-pyridylmethyl)-3-cephern-4-carboxylic acid betaines may, then beisolated by standard procedures of precipitation and crystallization.

Depending on the reaction conditions used, the new compounds of thepresent invention are obtained in the free form or in the form of theirsalts. From the salts it is possible to prepare the acids in knownmanner, or from the acids the salts are readily accessible, for example, by reaction with hydroxides, carbonates or bi- I to screen foractivity against pathogens. The antibacterial spectrum of typicalcompounds of the present invention, representing the concentrationrequired to inhibit the growth of various typical bacteria, wasdetermined in a standard manner by the agar-dilution streak-plate tech-Example 4.Prcpararin of 7-(5-azihexanoylamino)- cephalosporanic acid Inplace of the 4-azipentanoyl chloride of Example 1,

nique which is commonly used in testing new antibiotics. there isemployed an equimolecular quantity of S-azihexa- The following tablesummarizes the in vitro activity of noyl azide whereby the7-(S-azihexanoylamino)ceph- 7-(4-azipentanoylamino)cephalosporanic acid(1), 7-(3- alosporanic acid is obtained in equally good yield.azibutanoylamino)cephalosporanic acid (2),7-(5-azihexanoylamino)cephalosporanic acid (3), 7-(4-azi-2- Example5.Prep-aration of 7-(4-azihexan0ylam-in0)-phenylpentanoylamino)cephalosporanic acid (4) and 7- cephalosporanicacid (4-azi-6-carboxyhexanoylamino)cephalosporanic acid (5 as comparedwith Cephalosporin C (6) again t a i- In the manner described in Example1, treatment of ety of disease-causing microorganisms.7-aminocephalosporanic acid with p-nitrophenyl 4-azi- Minimal inhibitoryconc. (meg/ml.) Organisms Staphylococcus aureus No. 11 1.56 6.25 3.121.56 6.25 100 Staphylococcus aureus ATCC 13709 0. 78 1. 56 0.39 0.78 3.12 50 Streptococcus pyogenes 0-203 0.78 0.2 0. 39 3. 12 Bacillus ccreuaATOC 10702 3.12 1. 56 25 25 100 The compounds of the present inventionretained antihexanoate produces the 7-(4-azihhexanoylamino)cephbacterialactivity after exposure to cephalosporinase, a alosporanic acid.bacterial enzyme, which completely destroyed the ac- I p tivity ofCephalosporin C under the same conditions. Example i f isxgg g z Si 2gizienylpenmnoyl' The high in vitro antibacterial activity of the novelp p n c a 1 compounds of the present invention makes them useful Yreplacing the 4-azipelitahoyl chloride p y in as additives to materialswhich are subject to microbial EXaInPle 1 Wi h an equimolecular quantityof 4-azi-2- deterioration such as cutting oils and fuel oils. They arephehylpehtaheyl chloride and following Substantially the also useful insoaps, shampoos and topical compositions Same Procedure described inExample theie is Obtained for the treatment of wounds and burns. e 7 all2"PhehYlPehiahoylaihihe)eepliellesperelllie The invention will bedescribed in greater detail in 5 acid- COHJllIlCtlOIl with the followingspecific examples. 3 Example 7 (4 azi 6 cm.boxyhewnoyl Example1.Preparation of 7-(4-azipenfaw0ylamino)- mino)c phalos poranic acidcephaloiipommc acid The procedure of Example 1 is repeated, substitutingTh id hl id f 4- i t i id i prepared b an equimolecular amount of4-azipimelic acid dichloride reacting 25 6 mg. (2 mmole) of the acidwith excess oxalyl 40 for the 4-azipehialieyl C'hleiide employed in thatp chloride and vacuum-evaporating the excess oxalyl chlo- There is thusobtdined the y y ride. The acid chloride residue is dissolved in 5 ml.of P P C acidacetone and added dropwise to a stirred, ice-cooled solu-Example 8' Prepwmti0n of 7 (4 azipem.anoylamino) s Prepared by 548 (2mmole) of (l-pyridylm ethyl)-3-cep-hem-4-carboxylic acid betaineaminocephalosporanlc acid and 550 mg. (6.6 mmole) of sodium bicarbonatein 30 ml. of Water and 24 ml. of Q gram of the e i salt of p nylacetone. The reaction mixture is stirred for 2 hours inaIPmM'CePhaIOSPOFamC acid f 3 ml. of Py Was an ice bath. The acetone isvacuum-evaporated and the dlsolved m of Water, which Was j d to P 6residual aqueous Solution acidified to PH 2 with hydro with acetlc acid,and stored under nitrogen for 3 days at chloric acid. The solution isextracted three times with The Solution e evaporated to yn nd the 25milliliters each time f ethyl acetate which is then resldue wastriturated with acetone to give 230 mg. of dried over magnesium sulfate.Evaporation gives 480 mg. the P of7-(4-azipentanoylamino)cephalosporanic acid. The sodium salt is obtainedby slurrying this product in 10 (23 2 31 2 ml. of water and addingdilute sodium hydroxide dropp y p r oxy 16 6 wise to pH 5 to effectsolution. Vacuum-evaporation to one gram of the Sodium Salt of P 3 asmall volume and addition of a large quantity ofaceamin)eePhal0SPTahieacid and 8 1111- Of Pyridine Was tone precipitates440 mg. of sodium 7-(4-azipentanoylsolved in 50 ml. of Water, which wasadjusted to pH 6 with amino)cephalosporanate as a white solid. aceticacid, and stored under nitrogen for 3 days at 37 C- The solution wasevaporated to drynesss and the residue Example W 7 Was triturated withacetone to give the product.

cephalosporanic aczd By replacing the 4-azipentanoyl chloride employedin Example 10' Prepamtwn of 4'azlpenmnol'l Example 1 with anequimolecular quantity of 3-azibuta- A Solution of 154 0f4-ket0Petan0l-l in 50 0 noyl chloride and following substantially thesame promethanol is added carefully to 150 ml. of liquid ammonia ceduredescribed i E l 1 th i bt i d th 7- and the solution is stirred atreflux temperature for 5 /2 (3-azibutanoylamino)cephalosporanic acid.hsilTd'hre sollution isocooled in Dry Ice-acetone and 20 g. o y oxyamino- -su onic acid in 80 ml. methanol is Example hz of7'gs'azlgenmnoylammm' added over a period of about /2 hr. The colorlessmixcep a Ospommc act ture is warmed to reflux and stirred for 2 hrs.,then al- The procedure of Example 1 is repeated, substituting lowed towarm to room temperature overnight allowing an equimolecular amount of3-azipentanoyl bromide for for the evaporation of excess ammonia. Themixture is the 4-azipentanoyl chloride employed in that example.filtered, the precipitate is Washed with several small por- There isthus obtained the 7-(3-azipentanoylamino) tions of methanol, and thefiltrate and washings are comcephalosporanic acid. bined. The resultingsolution is evaporated at reduced pressure until no further ammoniaremains in the solution (about 6 original volume). This solution gives astrong positive test on acidic starch iodide paper. To this solution isadded 35 ml. triethylamine, the solution is cooled in ice, stirredrapidly, and a solution of iodine in methanol is added until the redcolor of iodine persists for several seconds. The resulting solution isevaporated and the residue is distilled to yield the product, B.P. 42 C.at 2.5 mm.

Example 11.-Preparation of 3-azibutan0l-1 In place of the4-ketopentanol-l of Example 10, there is employed an equimolecularquantity of 3-ketobutanol-1 whereby the 3-azibutanol-l is obtained inequally good yield.

Example 12.Preparatin of 4-azipentan0ic acid In the manner described inExample 10, treatment of 4-ketopentanoic acid with liquid ammonia andhydroxylamino-O-sulfonic acid produces the 4-azipentanoic acid.

Example 1.Preparati0n of 3-azibutan0ic acid To 12.0 ml. of a solution of26.72 g. of chromic acid in 23 ml. of concentrated sulfuric acid dilutedto 100 ml. with water is added drops concentrated sulfuric acid. Thesolution is cooled to 2 C. and 2.00 g. of 3- azibutanol-l is addeddropwise with stirring. The solution is stirred at 4 C. for 1 hr., thentreated dropwise with 20 ml. of 5 N sodium hydroxide solution. Theresulting precipitate is filtered off, the filtrate is saturated withsalt and extracted with several portions of ether. The aqueous phase ismade strongly acidic with hydrochloric acid and the resulting solutionis continuously extracted with ether for 5 hr. The ethereal extract isdried over sodium sulfate, and the ether is removed at reduced pressure,leaving an oily residue. Distillation of the residue at 49 5 1 C. at0.16 mm. affords the product.

Example 14.-Preparation of 4-azi-2-phenylpentanoic acid A stirredsolution of 20 g. of a-phenyl levulinic acid in 500ml. of liquid ammoniais stirred at reflux temperature for six hr., cooled to 45 C. and asolution of 20 g. of hydroxylamine-O-sulfonic acid in 100 ml. methanolis added over a period of 1 hr. The mixture is stirred at reflux for 1hr. and allowed to warm to room temperature overnight allowing excessammonia to escape. The mixture is filtered, the precipitate is washedwith methanol and the filtrate and washings are combined. To thissolution is added 20 ml. of 5 N sodium hydroxide and the solution isevaporated at reduced pressure to a volume of about 100 ml. The solutionis cooled to l5 C. and acidified to pH 3 using hydrochloric acid. Theacidic solution is extracted with several small portions each ofmethylene chloride and ether. The aqueous solution is made basic withsodium hydroxide and then added dropwise with ice cooling to a wellstirred mixture of silver oxide in water (made up by adding withvigorous shaking a solution of 200 ml. of 2 N AgNO to a solution of 200ml. of 2 N NaOH). The mixture is filtered through Celite, made stronglyacidic with hydrochloric acid an dextracted with several portions ofmethylene chloride. The organic extracts are combined and dried oversodium sulfate. After removal of the solvent at reduced pressure, thecrystalline residue is recrystallized from methanol-water to afford 9.54g. of 4-azi-2-phenylpentanoic acid, M.P. 73.5 75 C.

Example 15.Preparation of 5-azihexanoic acid 5-azihexanoic acid isprepared from S-ketohexanoic acid essentially by the method used for4-azi-2-phenylpentanoic acid of Example 14. The product isrecrystallized from 30-60 C. petroleum ether; M.P. 1416 C;

6. Example I6.Preparati0n 0] 4-a zi-6-carboxyhexanoic acid A solution of25 g. of 4-ketopimelic acid in 250ml. of liquid ammonia and 50 ml. ofmethanol is stirred for 5 hours'at reflux, cooled to 40 C. and 5portions of 4 g. each of hydroxylamine-O-sulfonic acid in 25 ml.methanol are added at 15 minute intervals. The mixture is stirred atreflux for about 1 hour, then allowed to warm to room temperatureovernight allowing excess ammonia to evaporate. The mixture is filteredand the precipitate is dried. One-fifth of the crude diaziridine issuspended in ml. of methanol and treated with 6 g. of sodium hydroxide.The mixture is shaken 15 minutes and the solvent is evaporated at 40 C.under reduced pressure to about 35 ml. The residue is added to a stirredmixture of silver oxide in methanol. (The silver oxide is prepared bymixing together 100 ml. of 1 N sodium hydroxide with a solution of 17 g.of silver nitrate in 100 ml. of water, allowing the silver oxide soformed to settle, decanting the supernatant solution and washing theresidual silver oxide by decantation with four portions of methanol ofabout 200 ml. each. The silver oxide is then suspended in 200 ml. ofmethanol.) The mixture is stirred for 72 hours, filtered through Celite,the filtrate retreated with a similar quantity of silver oxide at 50 C.for 6 hours, refiltered through Celite, and the filtrate is evaporatedunder reduced pressure to about 75 ml. The residual solution is madeacid to Congo Red paper with hydrochloric acid and the productcrystallizes, MP. 121 l23 C. The product may be recrystallized fromhexane with no change in the melting point.

What is claimed is:

1. A compound selected from the group consisting of those of theformula:

wherein in has a value selected from the group consisting of 0, 1 and 2;n has a value selected from the group consisting of 0, 1 and 2; A isselected from the group consisting of acetoxy and N-pyridiniurn; M isselected from the group consisting of hydrogen, pharmaceuticallyacceptable non-toxic cations and an anionic charge when A isN-pyridinium; Q is selected from the group consisting of hydrogen andcarboxy; and W is selected from the group consisting of hydrogen andphenyl with the proviso that when W is phenyl then n must not be 0; andthe non-toxic pharmaceutically acceptable basic salts thereof when Q iscarboxy.

2. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is hydrogen, n is 1, m is 0 and Q is hydrogen.

3. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is hydrogen, n is 0, m is 0 and Q is hydrogen.

4. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is hydrogen, n is 0, m is 1 and Q is hydrogen.

5. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is hydrogen, n is 2, m is 0 and Q is hydrogen.

6. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is hydrogen, n is 1, m is 1 and Q is hydrogen.

7. A compound according to claim 1 wherein A is acetoxy, M is hydrogen,W is phenyl, n is l, m is 0 and Q is hydrogen.

8. A compound according to claim 1 wherein A is 7 8 acetoxy, M ishydrogen, W is hydrogen, n is 1, m is 1 N-pyridinium, M is an "anioniccha'rge, W is hydrogen, and Q is carboxy. n is 1, m is 2 and Q ishydrogen.

9. A compound according to claim 1 wherein A is N- pyridinium, M is ananionic charge, W is hydrogen, n is NO references elted. m 15 0 and Q 18hydmgen- 6 NICHOLAS s. RIZZO, Primary Examiner.

10. A compound according to claim 1 wherein A is

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF THOSE OF THEFORMULA: